Optimization of Agrobacterium tumefaciens-Mediated Transformation of Aspergillus niger and Expression of Lipase

Abstract:

The method for Agrobacterium -mediated transformation of Aspergillus niger was improved by optimizing the co-culuture materials, Agrobacterium species, inducer concentration, co-culture time and temperature as well as the Agrobacterium concentration during co-culture. The optimum conditions for the transformation of A. niger were 10 ⁷ /mL non-germinated fresh spores and Agrobacterium with OD₆₀₀ 0.9~1.0 in a ratio of 1∶1, and 200 μmol/L acetosyringone in the IM plate. The mixed cells were then transfected at 23 ℃ for 48 h, and the number of transformants reached 60 ± 5 transformants / 10⁶ spores, and the positive rate reached over 90%. The expression vector of homologous A. niger lipase with constitutive or inducible promoter was constructed and transformed into A. niger by the optimized Agrobacterium-mediated transformation method. Finally the positive transformants were identified by Rhodamine-olive oil plate.